Process for preparing an antibiotic substance which comprises fermenting the organism streptomyces hazeliensis

ABSTRACT

PROCESS FOR PREPARING A COMPLEX OR MIXTURE OF ANTIBIOTIC SUBSTANCES INCLUDING A COMPOUND OF THE FORMULA   3,4-BIS((4-(HO-),7-((3-(HO-),4-((5-(H3C-)PYRROL-2-YL)-   COO-),5-(H3C-O-),6,6-DI(H3C-)PYRAN-2-YL)-O-),8-(H3C-)   CHROMEN-2-ON-3-YL)-NH-CO-)PYRROLE   WHICH COMPRISES FERMENTING THE ORGANISM STREPIOMYCES HAZELIENSIS (NRRL 2938) UNTIL ACTIVITY AGAINST GRAM-POSITIVE BACTERIA IS OBTAINED.

United States Patent Oifice 3,723,254

Patented Mar. 27, 1973 DETAILED DESCRIPTION PROCESS FOR PREPARING AN ANTIBIOTIC The organism WhlCh produces the novel antibiotics of STANCE WHICH CMPRIES FERMENTING THE the invention is named Streptomyces hazeliensis var. ORGANISM STREPTOMYCES gys hazeliensis nov. sp. This organism was isolated from a Julius Berger, Passaic, N.I., assignor to Hofimann-La 5 sample of soil obtained in Matane, Gaspe, Canada. The Roche Inc Nutley, NJ- organism will be designated hereinafter as Strepto myces gz g fgg fiog i i l ggg :Ig;;f ?g%%n ?6 ghazeliensis for purpose of brevity. A culture of Strepto- 5 656 653 y myces hazelzen-szs has been deposited 1n the collectlon of Int. C07: 47/18 m1croorgan1sms of the United States Department of Agri- Us. CL 4 Claims 10 culture, Northern Utilization Research and Development Division, Peoria, 111., under the Registration No. NRRL 2938. ABSTRACT OF THE DISCLOSURE The antibiotics of the invention are characterized by Process for preparing a complex or mixture of antihaving a high activity against Staphylococcus aureus. biotic substances including a compound of the formula Additionally, the antibiotics are active against a wide l 4 ll, up

which comprises fermenting the organism Streptomyces range of gram-positive bacteria, and gram-negative bachwzeliensis (NRRL 2938) until activity against gram-positeria, e.g., against A. aer genes, Mycobacteriumphlei, tive bacteria is obtained. Proteus vulgaris, Sarcinu lutea PC1-1001, etc.

Accordingly, the antibiotics of the invention are useful in the treatment of bacteria infections, for example,

RELATED CASES those caused by Staphylococcus aureus. They can be ad- This application is a division of United States patent ministered by parenteral injection, e.g. in a parenterally application Ser. No. 325,498, filed Nov. 11, 1963 in the acceptable vehicle or orally. names 0t Andrew David Batcho, Julius Berger, Andre The antibiotics of the invention have been named Furlenmeier, Oscar Keller, Benjamin Pecherer, Arno sugordomycins. The most active sugordomycin antibiotic Johannes Schocher, Hans Spiegelberg and Bruno Peter against Staphylococcus aureus is (I) N,N-bis{4-hydroxy- Vaterlaus. This application also discloses only the subject 8 methyl 7 [4-O-methyl-5,5-dimethyl-3-O-(S-methylmatter found in application Ser. No. 366,887, filed May 2 pyrrolylcarbonyl a L-lyxopyranosyloxy]-2-oxo-2H- 12, '1964, and now aabndoned, in the names of Andrew 1 benzopyran 3 yl} 3 methyl 2,4 pyrroledicarbox- David Batcho, Julius Berger, Andre Furlenmeier, Oscar amide. This compound has the structural formula CH5 0 l CH: CH; 0 -1 OCH; on, O K E: 01130 l 0 0 cm CONH CH: \l,

)i -NHCO u 6H H o N l] H CH; 0 H OH N I5 H o H Keller, Benjamin Pecherer, Arno Johannes Schocher, Hans Other antibiotics within the scope of the invention in- Spiegelberg and Bruno Peter Vaterlaus. clude the following:

CH3 0 CH3 CH3 0 OCH; on: I 0 K mo omo i*- o 0 CH3 -CO-NH K B: \L.

-NH-CO OH H 0 j N II 5 H 0-0 cm 0 OH H N g H 0 N H I) 2N-{4 hydroxy 8 methyl 7 [4-O-methyl-5,5-dimethyl- 3 0(2 pyrrolylcarbonyl) u L-lyxopyranosyloxy1-2- 2N{4 hydroxy 8 methyl 7[4-Oemethyl-5,5-dimethyl- 3 (2 pyrrolylcarbonyl a-Iflyxopyranosy1oxy]-2- 0x0 2H 1 benzopyr'an- 3 yl}-4N'-{4-hydroxy-8- methyl 7'[4 O-methyl-S,5-dimethyl-a-L-lyxopyranosyloxy] 2 0x0 2H-1-benzopyran-3-yl}-3-methyl-2,4- pyrroledicarboxamide 6 of Bacteria by the Committee on Bacteriologic Technic of the Society of American Bacteriologists, 1948; and by Waksman, The Actinomycetes (Chronica Botanica Co.,

On Bennetts and Czapeks agars, all strains of Streptomyces hazeliensis grew abundantly at 22, 28, and 35 2N{4 hydroxy 8 methyl-7[4-O-methy1-5,S-dimethyla L lyxopyranoslyoxy] -2-oxo-2H-1-benzopyran-3-yl}- 4N{4 hydroxy 8 methyl-7[4-O-methyl-5,5-dimethyl- 3 0(2 pyrrolylcarbonyl-a L-lyxopyranosyloxy]-2-oxo- 2H 1 benzopyran 3 yl} -v 3-methyl-2,4-pyrroledicarboxamide Streptomyces hazeliensis is an aerobic and aerially sporulating member of the order Actinomycetales and belongs to the genus Streptomyces as described in Bergeys Manual of Determinative Bacteriology, 7th edition, 1957. Although this organism may be considered related to Streptomyces griseoflavus strain No. 160, Streptomyces niveus NRRL 2446, Streptomyces spheroides NRRL 2449, Streptomyces No. 58383, Strp-tomyces spirogriseus NRRL 2590, and'Stre'ptomyces griseus ATCC 12318, there are more than sufficient. ditferences between the above organisms and Streptomyces hazeliensis to definitely determine that the latter is' not the same species as any of the former. The difierences in morphology and in spore color are clearly suflicientto differentiate Streptomyces hazeliensis from all of the antibiotic-producing Streptomyces according to the classification criteria of T. G. Pridham, C. W. Hesseltine and R. G. Benedict, Appl. Microbiology 6: 52-79 (1958) A guide for the classification of Streptomyces according to selected groups."

Another conclusive differentiation lies in the difference between the antibiotics produced by Streptomyces hazeliensis and novobiocin produced by the above organisms.

(hiltures of Streptomyces hazeliensis grown on favorable media, such as tomato-soy agar, at 28 C. for 2 to 10 days grow with an aerial mycelium which is generally white and becomes grey to grey-brown on sporulation. A colorless to yellow exudate is formed on various media.

Sporophores on Bennetts and asparagine agar are main- 1y straight to fiexuous, ending sometimes in a wide hook or loop, or in loose, open, characteristically wide (diameter 7y.) spirals with 1 to 5 loops, the spirals being quite irregular in shape and distance between the loops. The spores are oval to cylindrical, varying from 1 to 1.2, wide x 1.4 to 2.4g in length. No well-formed spirals were seen on either Czapek or yeast-malt agar media, but there were present small hooks or primitive spirals. Sometimes up to 5 sporophores arise from one point in a broom-like form. There are no ball-like, tight or compact spirals, thus differentiating the culture from Streptomyces spheroides. Al though some clusters of sporophores do occur, they are not characteristically corkscrew-coiled at the tip, as they are for Streptomyces niveus. On the contrary, the coils of a sporophore of Streptomyces hazeliensis are bigger at the tip, wider and looser, becoming narrower and tighter at the point of attachment to the hypha. v To provide a basis for comparing Streptomyces hazeliensis with known Streptomyces described in classical complex nitrogenous media, as well as on chemically defined media [Pridham and Gottlieb, I. Bact. 56, 107-115 (1948)], both types of media were used. Media and procedures were adapted from those described above by Pridham and Gottlieb; Gottlieb D., App. Microbiology 9: 55- 65 (1961); A Manual of Methods for Pure Culture Study Sucrose-nitrate agar (Czapek sucrose) Growth, good, but no sporulation; aerial mycelium creamy colored (between hue 2 cc. and chm grey scale (1). Reverse cream-brown; pigment creamy (hue ca. 2).

Glucose-asparagine agar Growth abundant, slightly raised, smooth surface aerial mycelium light grey, with light grey spores. Reverse pale cream; no soluble pigment. One strain shows tiny clear droplets of exudate at end of streaks.

Glucose-nutrient agar Growth scarce, flat, leathery, with strong foldings into agar. Aerial growth white, reverse dark cream; soluble pigment brownish (hue 4 ne). Streptomyces spheroides gives moderate growth with no soluble pigment, while Streptomyces niveus gives good growth and soluble yellow pigment. Streptomyces sp. No. 58383 gives abundant growth, no soluble pigment.

Glucose-nutrient plus glycerol agar Abundant growth, very deep folds into agar; surface wrinkled and cracked. Vegetative mycelium light tan, with some fairly large drops of light yellow colored exudate. Aerial growth partly white to greyish-brown (chm. near grey 5 dc), about 25 percent sporulated. Reverse coffee brown; soluble pigment light brown-yellow (hue 3 Le.) to brown.

Amidex agar Growth moderate to good, flat; aerial mycelium medium grey; reverse cream-brown to dark cofiee brown. Soluble pigment red-brown (hue 4 pe-pg).

Tomato paste-oatmeal agar Growth heavy, with good sporulation; some exudate droplets. Aerial mycelium grey to grey-brown (chm near grey 5 fe). Reverse dark brownish olive with dirty olive soluble pigment.

Skim milk agar Substrate growth good, pasty, smooth, with no aerial mycelium. Strong production of soluble dark brown pigment, light brown further away from the growth streaks (hue 5 n-i and hue 3 i.e).

Peptone-iron agar A few colonies only; smooth pastry, regularly ridged cone with bright white top. Aerial mycelium dark cream, top white. Soluble pigment dark brown (hue 4 pn).

Bennetts agar Growth abundant, aerial mycelium grey with greybrown (reddish) spores and production of droplets of pale yellow exudate. Reverse yellow-brown; soluble pigment light yellow-brown (hue 3 pc). A second strain gave no exudate, while a third gave a spreading yellow exudate which wetted the whole growth surface.

Potato plug Growth scant after 2 weeks, but blackening of potato after only 4 days. On the other hand, Streptomyces splzeroides and Streptomyces niveus both first gave scant growth, slowly becoming quite good, with greyish-white sporulation and brownish discoloration of potato. Sterptomyces No. 160 gives abundant, much wrinkled growth.

Carrot plug Very good substrate growth, white becoming smokegrey to blue-grey when sporulated. Some clear yellow exudate. Strepmmyces niveus gave moderate growth with white aerial mycelium, while Streptomyces spheroides gave no growth.

Proteolytic activity (a) Gelatin: Triplicate tubes were stab inoculated with a needle and spore suspension. Before judging, tubes were held 1 hour at 3 C., then returned to room temperature. In 7 days, no liquefaction, small white colonies on surface with dark brown soluble pigment (hue 2 pi-pl) immediately below the colonies. By 14 days, about 25 percent liquefaction, fair growth on surface plus some pellets submerged in liquified portion; dark brown soluble pigment. After 21 days, 100 percent liquefaction, color of gelatin brown-yellow (hue 2 pc) while color of control tubes was hue 1 /2 ca. Streptomyces niveus and Streptomyces spheroides both are reported to give good growth on gelatin but no soluble pigment. The former gives partial liquefaction and the latter rapid liquefaction (3 to 10 days). Streptomyces No. 160 gives rapid liquefaction, no soluble pigment. Streptomyces sp. No. 58383 gives poor growth on gelatin and no liquefaction.

(b) Milk: In 2 days, all five Streptomyces hazeliensis strains tested gave slight surface growth pellicle with creamy color; no coagulation or peptonization; pH by glass electrode same as control tubes, 6.0 to 6.2.

By 7 days, all give good firm surface growth pellicles, creamy to light brown color. Brown ring on tube Wall; soluble pigment cream-yellow to light orange. One strain shows light peptonization; pH range 5.3 to 5.7 (control pH 5.7-6.1).

After 14 days, surface growth good but no sporulation; aerial mycelium leathery, rose-cream to a slight brown or lilac; no coagulation but slight peptonization. Cream-orange color (hue 4 ca-en) in unpeptonized portion; pH 5.2 to 5.7 (control pH 5.6).

Streptomyces No. 160 gives strong coagulation and strong peptonization of milk.

Nitrate reductase activity (a) Organic broth: Growth good by 14 days, thick white pellicle; dark yellow soluble pigment. Slight nitrite production becoming strong after 21 days; heavy surface growth starting to sporulate, greyish.

(b) Inorganic broth: Light growth with no reduction for 14 days; good surface growth by 21 days, but no nitrite production, with nitrate still present. Smith et al., Streptonivicin, A New Antibiotic, Antib. and Chemother. 6, 135-142 (1956), find that Streptomyces niveu's gives no nitrate reduction on either synthetic or nutrient nitrate broth; Streptomyces spheroides gives only faint reduction of nitrate agar to nitrite in 4 days, while Streptomyces sp. No. 58383 gives strongly positive nitrite production.

Production of hydrogen sulfide After 21 hours, slants showed very faint growth but strong H S development; by 14 days, shiny looking colonies; dark blue color of medium changed to dark brown.

Tyrosine agar All 5 strains tyrosinase positive, with light growth in 14 days, aerial mycelium white, reverse white; no pigment after 7 days, but by 14th day, pigment is beige-grey, hue 3 ec-ge (control hue 1 /2 db).

Carbon utilization Xylose, arabinose, rhamnose, glucose, fructose, sucrose, lactose, rafiinose (i), inositol, cellobiose, trehalose, citrate, malate, and succinate are utilized. Not utilized are inulin, maltose, mannitol, sorbitol, acetate, formate, oxalate, and tartrate.

It is to be noted that Streptomyces niveus and Streptomyces spheroides have been found to utilize inulin, maltose, mannitol and sorbitol in contrast to Streptomyces hazeliensis, whereas they did not grow on the citrate or succinate media in parallel trials where Streptomyces hazeliensis did. Streptomyces niveus utilizes formate, oxalate, tartrate, and sodium citrate.

Nitrogen utilization L arginine is utilized. Not utilized are methionine, sarcosine, creatine, amino-isobutyric acid, taurine, and betaine.

Antagonistic properties Produces the sugordomycin antibiotics, but has not been found under any condition to produce novobiocin. Streptomyces spheroides and Streptomyces niveus both produce novobiocin but no sugordomycin antibiotic, even on media where Streptomyces hazeliensis yields only sugordomycin antibiotics.

In summary, Streptomyces hazeliensis differs morphologically both from Streptomyces mveus and Streptomyces spheroides in its lack of corkscrew coiled loops occurring at the tips of the sporophore (Streptomyces niveus) and its lack of tight, compact, ball-like spirals typical of Streptomyces spheroides. 1

While Streptomyces niveus and S treptomyces spheroides cause a characteristic cracking and splitting of nutrient agar media very early, Streptomyces lzazeliensis generally does not. Streptomyces niveus freqeuntly produces a soluble pigment on various agar media, whereas Streptomyces spheroides is generally not a pigment producer. Streptomyces hazeliensis, on the other hand, generally does produce pigment, even more than Streptomyces niveus.

It has also been discovered that the composition of the media in which the Streptomyces hazeliensis is cultured markedly affects (a) the amount of antibiotic produced, (b) the rate of antibiotic formation, and (c) the kind of antibiotic mixture in the broth.

The potency of fermented broths produced in flasks or tanks is determined by the usual cup-plate agar diffusion method, using Staphylococcus aureus as the test organism. (D. C. Grove and W. A. Randall, Assay Methods of Antibiotics. A Laboratory Manual, Medical Encyclopedia, Inc., New York 1955, pages 7-16.)

One unit of activity was arbitrarily assigned to that amount of antibiotic which when dissolved in 1 ml. of aqueous buffer produced an inhibition zone of 18 mm. diameter on the assay plate. The cylinders themselves are 8 mm. in diameter. The pure antibiotic I has a reference value of 5500 units/mg.

Green or yellow split peasconstitute a superior source of nitrogen. Soybean flour, corn distillers dried grains with solubles, cottonseed flour, coconut protein meal, linseed oil meal, anddried corn fermentation solubles with grairHSoluferm)"all-"gave fair to good yields, which were usually inferior,""however, to pea-containing media. Theaddition to pe'as of small quantities of ferrous sulfate, bone-ash, or cornsteep dried solids did not increase antibiotic yields; but the presence of CaCO and K HPO did result in'an' increase in antibiotic yields.

The addition of a carbohydrate to natural complex nitrogen sources had variable eflFects; certain carbohydrates proved to be harmful to antibiotic production. With certain othercarbo'hydrates,the carbon/nitrogen ratio in the medium must be controlled in order to obtain a good yield of antibiotic. The addition to a yellow pea medium of 0.25'p'ercent levels of sodium salts of organic acids all caused the inhibition of antibiotic production. In this experiment a basal medium was used containing 3 percent yellow split peas, 0.1 percent CaCO and 0.1 percent K HPO4.

Antifoam agents are generally employed to control the foaming produced during large fermenter runs. It was found that any of a variety of plant oils such as safflower, cottonseed, sesame, coconut, soybean, or animal oils such as lard, oleic acid, etc., can be used without decreasing antibiotic yield. Also, silicone antifoams did not decrease antibiotic yield.

In the following table the yield of antibiotic produced on the same media by Streptomyces hazeliensis, Streptomyces niveus, and Streptomyces spheroides is compared. It should be noted that all of the inhibition zones produced by the active Streplomyces niveus and Streptomyces spheroides media were neither clear nor sharp, whereas the zones produced by Streptomyces hazeliensis were very clear and sharp. These diiferences in appearance of the inhibition zones also serve to differentiate sugordomycin from novobiocin.

TABLE 3 Staphyloccua aureus plate units/m1.

4 days 6 days Strep. Strep. Strep. Strep. hazeli- Strep. spherhazeli- Strep. spher- Composition oi medium, in percent ensia 'm'veus oz'des 87181'3 m'veus aides 2 split peas, 1 cornstarch, 0.1 08003, 0.1 KrHPOl 69 50 47 320 53 5 4 cornsteep liquor, 5 glusoce, 0.05 KHgPO, 0.3 NaN O3, 0.1 MgSO.;- IH2O- O 0 0 Trace 9 2 soyflour, 2 brown sugar, 0.25 cornsteep dried solids, 0.]. K HPO4 Trace 0 0 13 0 0 2 distiilers dried solubles (Soludri) 2 glucose, 0.1 K2HPO4, 0.1 021003.- 0 54 37 0 69 2.5 Pharmamedla, 2i5fig e 0 48 0 3 Trace 2 Fermamine 1 type 1 starch, 0.1 K2HPO4, 0.05 MgSOa 0 8 19 0 6 6 Pharmamedia is a 56 Fort Worth, 'lexas.

percent protein-24 percent carbohydratecontalning material from cottonseed meal. Traders Oil Mill Cc,

. 2 Fermamine is an enzymatic protein hydrclysate of Sheffield Chemical 00., Norwich, N.Y.

The effect, of carbohydrate addition on antibiotic productiori'canlbe seenj'from the table given below in which the basal medium contained 2 percent yellow split peas.

" "TABIJEI S. aureaus units/ml. of

Addition. tobasal medium: Whole broth in 7 days l percentfcorn starchfl(coritrol) 180 '1 'p'ercent mannitol e 120 -1 I percent glycerol 50 "2"per'cent glycerol Trace 1 percent maltose 55 1 percent arabinose'. Q. 4

' -'1 percent rhamnose v 12 1 percent [lactose 18 1 percent inositol 3 in the following experiments recorded in Table 2, 0.1 percentCaCOgand 0.1 percent K HPO are present in addition to 2 percent yellow split peas to form the basal medium.

TABLE 2" 1 percent xylose...

Effect of mineral elements It has already been noted above that the addition of 0.1 percent CaCO and K HPO results in the production of higher yields of antibiotics. If both CaCO and K HPO are omitted or theconcentrations of either one or both are increased"to the0.5 percent level, drastic decreases in antibiotic yield result. Also, the addition of 0.3 percent concentrations of NaCl or (NH S O drastically decreases the antibiotic yield.

Accordingly, the process for producing a mixture of sugordomycin antibiotics comprises culturing Streptomyces hazeliensis in a nutrient medium containing organic carbon and nitrogen sources, preferably split peas (which is both a carbon and a nitrogen source), and optionally, though preferably, from about 0.05 to about 3 percent; preferably about 0.1 percent CaCO from about 0.01 to about 0.5 percent, preferably about 0.1 percent K HPO and from about 0.1 to about 3 percent, preferably from about 1 to about 2 percent of a carbohydrate, e.g. a starch or any sugar listed in C01. 8. The culturing is preferably carried out for from about 3 to about 8 days at an aeration rate of from about 0.5 to about 10 c.f.m. per 50 gallons of medium, and at a temperature of from about 20 C. to about 35 C. The antibiotic mixture is then isolated from the resulting fermentation broth.

The antibiotic mixture can be isolated from the fermentation broth by several techniques. One technique is to acidity the broth, preferably with phosphoric acid, although other mineral acids, e.g., I-ICl, H etc., can be employed, to a pH in the range of about 2 to about 7, preferably at a pH of about 4 to about 4.5. A filter aid, e.g., a diatomaceous earth, is then optionally added, and the acidified broth filtered. The filter cake thus obtained is suspended in an organic solvent, e.g., a lower alkanol, such as methanol, ethanol, butanol, isobutanol, etc., acetone, methylisobutylketone, butyl acetate, or mixtures of these solvents, e.g. acetone-methylene chloride, methanolbenzene, etc., with butanol preferred. The resulting slurry is then filtered or centrifuged and the clear filtrate or extract is neutralized with an alkali, e.g., 50 percent sodium hydroxide solution, to a pH of about 6 to about 7, and the solvent removed, e.g. under reduced pressure and at a temperature below about 50 C'., to form a concentrate, for example, to a concentration of about 4 of the original volume. This concentrate is then mixed with a nonpolar organic solvent, e.g., a hydrocarbon solvent or an ether, such as petroleum ether, B.P. 6090 C., benzene, hexane, pentane diethyl ether, etc., whereupon the crude antibiotic mixture precipitates in the form of its sodium salt or a mixture of its sodium salt and free form,

in which form it is readily recoverable, e.g. by settling, filtration, centrifugation, etc.

Another method that can be used to isolate the crude antibiotic from the fermentation broth is to filter olf the cells, acidify the filtrate with a mineral acid to a pH of about 1 to about 7, and then contact the acidified filtrate with a water-immiscible solvent such as butanol, butyl acetate, ethyl acetate, ether, chloroform, methylisobutylketone, etc. After separating the aqueous layer, the waterimmiscible solvent is removed to leave the crude antibiotic mixture, or alternatively, the water immiscible solvent solution can be passed through an adsorbent, e.g., an ion exchange resin, e.g., Zeo-Karb H, Dowex-SO W, Amberlite IRC50, Amberlite IR-4, Amberlite IR-lOO, Ionac A-300, Dowex-l chloride, Dowex-l base, Dowex-l formate, etc., or activated charcoal, etc., to adsorb the antibiotic mixture, which is then removed from the adsorbent by elution with a solvent such as methanol. The cells which were filtered off are then extracted with an organic solvent such as a lower alkanol, e.g., methanol, ethanol, butanol, isobutanol, etc.; acetone; butyl acetate; acetone-methylene chloride; methyl-isobutylketone; meth ylene-benzene; etc. The solvent is then removed leaving behind the crude antibiotic mixture, which can then be combined with the antibiotic mixture obtained from the filtrate above.

The crude sodium salt of the antibiotic mixture obtained above or the crude acidic antibiotic is purified by suspending the antibiotic mixture in acetone and treating it with sufiicient mineral acid, e.g., HCl, to give an acid pH, e.g., a pH of from about 2 to about 6, preferably about pH 4.0. The insoluble material is filtered off and the filtercake washed with a small quantity of acetone. The combined filtrate and wash are evaporated under vacuum to form a concentrate, e.g., about the original volume, and the concentrate added to water and benzene giving a suspension of solids in the two liquid phases. The suspension, which is the free acidic antibiotic mixture, is filtered off, the precipitate suspended in warm methanol, and the methanol suspension cooled. The solid which separates is filtered off. This solid is the purified free acidic antibiotic mixture.

The crude antibiotic mixture can also be purified by the preparation of its benzylamine salt. The antibiotic mixture isolated above can be dissolved in an organic solvent, e.g., n-butanol, n-butyl acetate, ether, ethyl acetate, etc., and treated with an excess of benzylamine. The benzylamine salt of the antibiotic mixture precipitates. Many of the impurities contained in the crude antibiotic mixture remain behind in the organic solvent The precipitated benzylamine salt is filtered off and the free antibiotics obtained from the benzylamine salt by treatment with an aqueous mineral acid, e.g., aqueous hydrochloric acid, followed by extraction of the free antibiotic mixture from CHaO 4-benzyloxy-7a-(2,3-0-carbonyl)- noviosyloxy-S-methyl-coumarin WUJ the aqueous system by the use of an organic solvent immiscible or only slightly miscible with water, such as n-butyl acetate, n-butanol, ether, ethyl acetate,.etc. The immiscible solvent is then removed and the residue reprecipitated, e.g., from a hot methanol-benzene mixture.

Other amines were employed in an attempt to purify the antibiotic mixture but only the benzylamine salt was found to 'be fully satisfactory. Among the other bases that are not satisfactory are calcium hydroxide, procaine, ethylenediamine, diethanolamine, monoethanolamine, N,N-dibenzylethylenediamine, dimethyleth'anolamine, hydroxyethylethylenediamine, diisobutylamine, and furfurylamine.

The crude antibiotic mixture can also be purified by distributing the crude mixture between an organic solvent, e.g., butyl acetate, ether, or mixtures of chloroform and isopropanol, etc., and an aqueous buffer solution buffered to about pH 6.8 to about 11. The antibiotic mixture is in the aqueous buffer solution, from which it can be isolated by acidification, e.g. with a mineral acid such as HCl, H etc., to about pH '2 toabout pH 5 and the resulting suspension is extracted with an organic solvent immiscible or only slightly miscible with water, e.g., nbutanol, n-butyl acetate, ether, ethyl acetate, etc. and the purified acidic antibiotic mixture is recovered from the organic solvent, e.g. by evaporation of the solvent.

The above antibiotic mixture purified by any one of the above techniques can then be separated into its constituent antibiotics, preferably by the use of a countercurrent distribution system, e.g., a Craig countercurrent distribution system. The organic solvent is an organic solvent immiscible or only slightly miscible with water, e.g., n-butanol, n-butyl acetate, ether, ethyl acetate, chloroformisopropanol, etc. The aqueous phase is water buffered to a pH in the range of about 6 to about 9, preferably about 8 to about 8.9. The bufiers that can be employed are those commonly used for this purpose, such as phosphate and borate buffers.

Alternatively, the purified antibiotic mixture can be used therapeutically as such, e.g., against Staphylococcus aureus by introducing the antibiotic mixture into the animal body, e.g., by injection or orally either in the acidic form or as a salt, such as the sodium salt, potassium salt or any other pharmaceuticallyacceptable base addition salt.

Another feature of the instant invention is the synthetic preparation of the most active antibiotic of the invention against Staphylococcus aureus, i.e. N,N-bis{4- hydroxy 8 methyl-7-[4-O-methyl-5,5-dimethyl-3-O-(5- methyl 2 pyrrolylcarbonyl)-a-L-lyxopyranosyloxy1-2- oxo-2H-l-benzopyran-3-yl} 3 methyl-2,4-pyrro1edicarboxamide. v

The synthesis of the above antibiotic'is carried out according to the following reaction scheme:

noviosyloxyi-metl1yl-eoumariu CHa O-[5-methyl-2'-carbony1-pyrrolelnoviosy1oxy-8-methyl-coumarin cm g methylmumarln l CHAKE'EO c g it, Am mc-LJ-Z=0 XIII XIV 3-benzenedlazo-4-hydroxy-7a-2-0- 3-amino-4-hydroxy7a-2-0- acyl-IiO-[H-methyl-W- carbonyl-pyrrolel-noviosyloxy-8- methyl-coumarin oxy-8-methyl-coumarin 0 OX X0 0 N H 2,4-diha1ocarbonyl-3- CH; 0 onto i'2i o 0 0 CH2 CONE l JNH-COlJ l H on H oni j-c=o H N,N' bis{4-hydroXy-8-methyl-7-[4-O-methyl-5,5-dimethyl-3-O (5-methyl 2 pyrrolylcarbonyl)-L-1yxopyranosyloxyl1-2-oxo 2H 1-benzopyran-3-yl}-3-methy1-2,4- pyrroledicarboxamide.

In the above reaction scheme, in the acyl group in Formulae XII and XIV the R group therein can be an alkyl group, an aralkl group, a phenyl group, or an alrkylor halo-substituted phenyl group, preferably a lower alkyl group.

The process of the invention shown in the above reaction scheme is carried out by (a) Reacting 4 benzyloxy-7'a-(2,3-O-carbonyD-noviosyloxy-S-methyl-coumarin IX) with a 2-methyl-pyrrole Grignard reagent, i.e. a Z-methyl-pyrrole magnesium hamethyl-pyrrole (X=Gl or Br) 15 where R has the meaning given above, e.g. acetic anhydride in pyridine to form 3-benzenediazo-4-hydroxy-7a-2- O acyl-3-O-[5-methyl-2-carbonyl-pyrrole] -noviosyloxy- S-methyl-coumarin (XIII). Other acylating agents that can be used to form compound XIII include the acyl halides, e.g., acetyl chloride, etc.

(e) Compound XIII is then treated with hydrogen in the presence of a hydrogenation catalyst, e.g., palladiumon-charcoal or other hydrogenation catalysts as given above for the hydrogenation of compound X to yield 3- amino-4hydroxy-7a-2-O-acyl-3-O ['methyl-2'-carbonyl-pyrrole] -noviosyloxy-S-methyl-coumarin (XIV) (f) Compound XIV is then reacted with a 2,4-dihalocarbonyl-3-methyl-pyrrole, wherein halo is chlorine or bromine, to give the antibiotic prepared by the process of the invention.

The invention will be better understood from a consideration of the following examples which are given for illustration purposes only and are not meant to limit the invention.

EXAMPLE 1 Streptomyces hazeliansis is stored in the spore form, in medicine bottles in either tomato-oatmeal agar, or in the lyophilized form, i.e., spores lyophilized in percent bovine albumin or 10 percent skim milk. An aqueous suspension of spores is inoculated into a six-liter shake flask containing 3 liters of medium composed of 1 percent ground split peas, 1 percent cornstarch, 0.1 percent CaCO 0.1 percent K HPO and 0.1 percent lard oil, and incubated at 28 C. for 3 to 5 days on a rotary shaker at 200 rpm. until good growth has occurred. The incubation is carried out in equipment which provides for aeration of the medium such as the above shake flask or an aerated bubbler bottle. The vegetative growth obtained is inoculated into a 100 gal. fermenter containing 60 gallons of a medium composed of 3 percent split peas, 0.1 percent CaCO 0.1 percent K HPO 0.03 percent silicone paste (Dow-Corning Silicone A paste), and 0.1 percent of lard oil. Alternatively, 60 gallons of the medium described above can equally well be employed. The fermenter is maintained at 28 C. for from 3 to 4 days with an airflow rate of 8 c.f.m. During this time the viscosity increases markedly and the pH drops to 5.96. 6. The above growth is then transferred to a 9000 gallon fermenter which contains 6000 gallons of sterilized medium composed of 3 percent ground split peas, 0.1 percent CaCO 0.1 percent K HPO and 0.03 to 0.05 percent of Dow-Corning Silicone A suspended in 0.1 percent of soybean oil. The fermentation is allowed to proceed at 28 C. for from 5 to 8 days depending on the vitro bioassay. Aeration is maintained at the rate of 25 c.f.m. of air for the first 24 hours, then 100 c.f.m. for 2472 hours, and 150 c.f.m. for 72-168 hours. At harvesting the whole broth is acidified to pH 3.9 by the addition of 85 percent sirupy phosphoric acid, followed by 1650 lbs. of a diatomaceous filter aid, and the acidified broth filtered. The filter cake is added to 2000 gallons of n-butanol with vigorous agitation. The butanol slurry is allowed to stand overnight without agitation, then centrifuged. The clear butanol extract is neutralized with 1482 ml. of 50 percent NaOH solution to about pH 7, and then flash evaporated under reduced pressure at a temperature below 50 C. to a volume of 50 gallons.

This concentrate is added to 250 gallons of vigorously stirred petroleum ether (B.P. 60 90") whereupon a precipitate of the crude sodium salt of the antibiotic mixture separates. This crude sodium salt probably consists of a mixture of the acidic and partially neutralized forms of the various components of the antibiotic mixture. The crude sodium salt is isolated by centrifuging and then dried.

EXAMPLE 2 Purification of the crude sodium salt (a) By distribution between solvent and buffers.50

g. of the above crude sodium salt is suspended in 1 liter of methanol and stirred for 30 minutes. The insoluble material is then filtered off and the filtrate concentrated in vacuo to a thick sirup. The thick sirup is suspended in 25 ml. of methanol and 625 ml. of 0.1 N hydrochloric acid. This suspension is extracted three times with 1.5 liter portions of ether and the combined ether extracts totaling 4.24 liters are extracted twice with 1.1 liters of pH 6.2 M/15 phosphate buffer, then twice with 1.1 liters of pH- 7.0 M/ 15 phosphate bufler. Little activity is extracted by this treatment. The ethereal solution is extracted twice with 1.1 liters of pH 7.8 M/ 15 phosphate buffer, whereupon the antibiotic mixture moves into the aqueous phase. The pH of this extract is lowered to 7.0 and the suspension extracted with n-butanol which dissolves the precipitate. The n-butanol is evaporated in vacuo leaving a solid. The solid is suspended in pH 6.5 M/15 phosphate buffer and the insoluble material, the purified antibiotic mixture, filtered off and dried.

(b) By preparation of the benzylamine salts.One hundred g. of crude sodium salt is dissolved in 10 liters of M/ 15 disodium phosphate butler of pH 9.5. The insoluble material is filtered ofl with the use of a filter aid and the dark filtrate acidified to pH 2.5 with concentrated bydrochloric acid. This suspension is extracted with an equal volume of butyl acetate which dissolves the precipitated antibiotic. The butyl acetate solution is then concentrated in vacuo to 1800 ml., 3. small amount of insoluble matter filtered off, then ml. of benzylamine dissolved in 100 ml. of butyl acetate added. A fine precipitate of the benyzlamine salt of the antibiotic separates. After settling, the precipitate is collected by filtration, washed first with a little butyl acetate, then anhydrous ether.

Twenty g. of a benzylamine salt of the antibiotic is dissolved in ml. of methanol, ml. of water added, and the pH of the suspension adjusted to 2.5 with hydrochloric acid. Another 300 ml. of water is then added to increase the fluidity of the suspension, and the mixture extracted twice with 400 ml. portions of butyl acetate. The combined butyl acetate extracts are concentrated to approximately 800 ml., at which point the crude acid has precipitated. The solid is filtered olf and washed with cold methanol. This product is then reprecipitated by dissolving it in hot methanol and benzene from which the antibiotic separates as a finely divided solid that melts at 228- 230 when dry.

(c) By acetone extraction.50 g. of the above crude sodium salt is suspended in 600 ml. of acetone, 30 ml. of 10 percent hydrochloric acid added and the mixture stirred overnight. The insoluble material is filtered off and the filtercake washed with a little acetone. The combined filtrate and wash is evaporated in vacuo to about 100 ml. To this concentrate are added 500 ml. of water and 200 ml. of benzene giving a suspension of solids in 2 liquid phases. The precipitate which is obtained from this suspension is the crude acidic antibiotic mixture. This precipitate is suspended in 100 ml. of methanol at 4050, cooled to room temperature, and then kept in a refrigerator overnight. The solid which separates is the purified antibiotic mixture.

EXAMPLE 3 Separation of the purified antibiotic mixture into its components 5 g. of purified antibiotic mixture obtained by the acetone purification process (c) is distributed in a 200-tube Craig countercurrent distribution apparatus between an upper aqueous phase of M/l5 K I-IPO butfer at pH 8.2 and a lower organic phase consisting of equal parts of isopropyl alcohol and chloroform. After equilibration of the buffer and organic phases, the distribution is carried out in the usual manner over the 200 tubes, each of which contains 40 ml. of each phase. The distribution gives a mixture of antibiotics I, II, III and 1V in tubes 1-140, and a mixture of antibiotics V, VI, VII and VIII in tubes 141- 200.

The run is then extended through an additional 200 tubes, this time with the buffer at pH 8.9.

In the upper phase of the extended run (total, 400 tubes), antibiotic I [identified above] is present in tubes 120 through 152. Antibiotic II or III is present in tubes 161 through 170. Antibiotic III or II is present in tubes 185 through 195. It is not known for certain which antibiotic designated II or III is present'in tubes 161 through 170 andwhich antibiotic is present in tubes 185 through 195. Tubes 153 through 160 contain a mixture of antibiotic I and either II or III and tubes 171 through 184 contain a mixture of antibiotics II and III.

In a lower phase, the tubes contain the same antibiotic distribution as in the upper phase.

The antibiotics are isolated from the solvent phase by combining the contents of the groups of tubes given above, e.g., for the preparation of antibiotic I tubes 120 through' 152 are combined, and the solvent removed by evaporating to dryness. For the buffer phase the contents of the groups of tubes given above are acidified to pH 2.5, the antibiotic extracted into butanol, a CHcl -isopropyl alcohol (1:1) solvent, and the solvent extract evaporated to dryness leaving the antibiotic as a residue.

Antibiotic I obtained from the above countercurrent distribution is then passed through another 200 tubes. Three isomers, obtained at tubes 90 through 100, at tube 120, and at tube 140 are obtained, indicating the presence of several closely related isomers, the absolute structures of which are not known at present. All of these isomers are bioactive, e.g., exhibit a high degree of activity against Staphylococcus aurews.

The mixture of antibiotics V, VI, VII, and VIII isolated above is resolved into a mixture of antibiotics V and VI, and a mixture of antibiotics VII and VIII by countercurrent distribution in a Craig countercurrent apparatus of 200 tubes. The solvent system employed consists of equal parts of the same organic phase employed above, i.e., an equal mixture by volume of isopropyl alcohol and chloroform while the aqueous phase is M/l5 K HPO buffer at pH 7.0. The solvent and buffer are equilibrated and the above antibiotic mixture (V-VIII) introduced into the apparatus and passed through the 200 tubes. The mixture of antibiotics V and VI is centered about the 110th tube and the mixture of antibiotics VII and VIII is centered about the 170th tube.

EXAMPLE 4 Preparation of 4-benzyloxy-7a-3-O-[5-methyl-2'-carbonyl-pyrrole]-noviosyloxy-S-methyl-coumarin (X) To a solution of CH MgI (prepared from 1.25 g. of Mg+3 cc. of CH I in 50 cc. of abs. ether) is added dropwise 8 g. of Z-methyl-pyrrole in 30 cc. abs. ether. The solution is refluxed for 10' minutes and added slowly to a solution of 4.8 g. of 4-benzyloxy-[2,3-O-carbony1-u-noviosyloxy]-8-methyl-coumarin (IX) in 200 cc. of abs. benzene. A precipitate forms immediately. The mixture is stirred for 30 minutes and then dilute HCl is added carefully. After addition of ethyl acetate the organic layer is washed successively with H O, dilute KHCO H dried over sodium sulfate and the solvent distilled off in vacuo. The residue, 4-benzyloxy7ot-3-O-[5'-methyl2carbonylpyrrole] noviosyloxy S-methyl-coumarin, is crystallized from benzene. It is recrystallized from acetonitrile; M.P'. 131132 (dec.).

EXAMPLE 5 Preparation of 4-hydroxy-7a-3-O-[5'-methyl-2'-carbonylpyrrole]-noviosy1oxy-8rnethyl-coumarin (XI) 4 g. of 4 benzyloxy 7u-3-O-[5'-methyl-2'-carbonylpyrrole] noviosyloxy 8 methyl-coumarin (X) are dissolved in 200 cc. of ethyl acetate and hydrogenated after addition of 2 g. of Pd on charcoal. 170 cc. of H are used (theoretical amount 150 cc.). After filtration the solution is evaporated to dryness; 3.2 g. of a yellowish compound are obtained. A small sample is crystalized for analysis from acetonitrile; M.P. 173-175 (dec.).

EXAMPLE 6 Preparation of 3 benzenediazo 4 hydroxy-7a-3-O- [5- methyl 2' carbonyl pyrrole]-noviosyloxy-8-methylcoumarin (XIII) 1.6 g. of aniline are dissolved in 120 cc. of H O+3.2 cc. of cone. I-ICl and diazotized at 5 with 1.1 g. of NaNO dissolved in 10 cc. of H 0. 10 minutes after the addition of the nitrite is complete, a solution of 6.4 g. of sodium acetate in 30 cc. of H 0 is added. 5 minutes thereafter a solution of 3.2 g. of 4-hydroxy-7a-3-O-[5'-methyl- 2'-carbonyl-pyrrole]-noviosyloxy-S-methyl-coumarin (X1) in 40 cc. of alcohol is added dropwise with stirring at 5. A yellow precipitate forms. Stirring is continued for three hours at 5. The precipitate is then extracted into ethyl acetate, washed successively with H O, I N HCl, H O, the solution dried over sodium sulfate and the solvent distilled off in vacuo. The product, 3-benzenediazo-4-hydroxy 7m 3 O [5-methyl-2-car-bonyl-pyrrole]-noviosyloxy-8-methyl-coumarin (XIII), is crystallized from benzene; M.P. 174-176".

EXAMPLE 7 Preparation of 3-benzenediazo-4-hydroxy-7a-2-0-acetyl-3- O [5' methyl 2'-carbonyl-pyrrole]-noviosyloxy-8- methyl-coumarin (XIII) 6.5 g. of 3-benzenediazo-4-hydroxy-7a-3-O-[5-methyl- 2. carbonyl pyrrole] noviosyloxy8methyl-coumarin (XII) is dissolved in 40 cc. of acetic anhydride and 20 cc. of pyridine and kept 18 hours at room temperature. The excess acetic anhydride is then decomposed with water, the resulting solution extracted with ethyl acetate and the ethyl acetate solution washed with l N HCl and with H O. After drying over sodium sulfate, the solvent is distilled off in vacuo. The residue, 3-benzenediazo-4- hydroxy 7a 2 O-acetyl-3-O-[5-methyl-2-carbonylpyrrole]-noviosyloxy-8-methyl-coumarin (XIII), is crystallized from isopropanol; M.P. 151-153 (dec.).

EXAMPLE 5 Preparation of 3-amino-4-hydroxy-7a-2-O-acetyl-3-0[5'- methyl 2 carbonyl-pyrrole]-noviosyloxy-8-methylcoumarin (XIV).

5.6 g. of 3-benzenediazo-4-hydroxy-7a-2-O-acety1-3-0- [5 methyl 2'-carbonyl-pyrrole]-noviosyloxy-8-methylcoumarin (XIII) are dissolved in 250 cc. of abs. alcohol. 1 g. of palladium on charcoal is added and the substance hydrogenated. 410 cc. of H are used (theoretical amount 400 cc.). After filtration the solution is evaporated to dryness, the residue dissolved in ethyl acetate and the solution washed with 0.5 N HCl and with water. After drying over sodium sulfate the solvent is distilled off in vacuo. There remains 5.3 g. of a yellow-brown foam, 3-amino- 4 hydroxy 7oz 2-O-acetyl-3-O-[5-methyl-2'-carbonylpyrrole] -noviosyloxy- S-methyl-coumarin (XIV) EXAMPLE 9 Preparation of N,N' bis{4 hydroxy 8 methyl-7-[4-O- methyl 5,5 dimethyl 3 O-(5-methyl-2-pyrrolylcarbonyl) L lyxopyranosyloxy] 2-oxo-2H-l-benzopyran-3-yl}-3-methyl-2,4-pyrro1edicarboxamide 5.3 g. of 3 amino 4-hydroxy-7a-2-O-acety1-3-O-[5- methyl 2' carbonyl -pyrrole]-noviosyloxy-8-methylcoumarin (XIV) is dissolved in 50 cc. of abs. pyridine. With stirring and cooling, 700 mg. of 2,4-dichlorocaribonyl-3-methyl-pyrrole, dissolved in 20 cc. of abs. tetrahydrofuran, is added dropwise and the solution kept at room temperature overnight. The dark solution is then diluted with ethyl acetate, washed successively with 1 N H and water, dried over sodium sulfate and the solvent distilled off in vacuo. There remains 5.6 g. of a brown foam. The latter is dissolved in about 80 cc. of liquid ammonia, which is slowly evaporated. The residue is dissolved in water and a small amount of alcohol, and the solution washed thereafter with ethyl acetate. Upon acidification with dilute H PO the precipitate is extracted with ethyl acetate. The solution is washed with Water, dried over sodium sulfate and the solvent distilled off. The residue, a brown foam, is crystallized from methanol/ benzene; M.P. 234-235 After recrystallization from acetonitrile/acetone the antibiotic, N,N-bis{4-hydroxy-8- methyl 7 -[4 O methyl-5,5-dimethyl-3-O-(5-methyl-2- pyrrolylcarbonyl) a L lyxopyranosyloxy]-2-oxo-2H- 1 benzopyran 3 y1}-3-methyl-2,4-pyrroledicarboxamide (I), shows a decomposition point of 238-239 (dec.).

A small sample is recrystallized from ethyl acetoacetate. DMF/H O; M.P. 258 (dec.).

The above antibiotic has a decomposition point which is difficult to establish exactly and is furthermore dependent upon the rate of temperature rise and apparatus.

What is claimed is:

1. The process for the production of an antibiotic substance which cornprises cultivating a strain of Streptomyces hazeliensis in an aqueous carbohydrate solution containing a nitrogenous nutrient under submerged aerobic conditions until substantial activity versus Gram-positive bacteria is imparted to said solution and then recovering said antibiotic substance from said solution.

2. The process of claim 1 in which the organism is Streptomyces hazeliensis NRRL 2938.

3. A prOcess according to claim 1 wherein the antibiotic substance is separated from the fermentation broth by extraction at an acidic pH in the range about 2 to about 7 of said antibiotic with a water-immiscible solvent in which said antibiotic is soluble.

4. A process according to claim 1 wherein the antibiotic substance is separated from the fermentation broth by extraction at an acidic pH in the range about 2 to about 7 of said antibiotic into a water-immiscible solvent in which said antibiotic is soluble followed by concentration of said antibiotic-containing solvent.

References Cited UNITED STATES PATENTS 3,201,386 8/1965 Kawaguchi et al 260-210 ELBERT L. ROBERTS, Primary Examiner J. R. BROWN, Assistant Examiner US. Cl. X.R.

UNITED STATES PATENT OFFICE @ERTIMCATE OF (ZURREUHQN Patent No. 397 39 5 Dated March 27, 1973 lnvent r( Julius Berger It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 1 Formula I should be FpRM Do-1050 (O-69) uscoMM-Dc suave-ps9 9' us. GOVERNMENT Pmrirmc orntz; min 0-3564,

V on 5x, a

UNTTED STATES PATENT ()FFTCE UERTTT TQATE T QURRECTTUN Patent No. 39725925 Dated Inventor(s) It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 1, line 42 "aaflmdonec1 should be abandoned Column 3 and a Formula V n CH3 OCHS CH should be OH OH Column 3 and Formula VI H 'n- -.a

1 FORM 8 0-1 (1 1 USCOMM-DC 60376-F'69 L \v U.S. GOVERNMENY PRINTING OFFICE: 959 -366-334.

TJNTTED STATES PATENT oTTTcE @ER'MNCATE 0F CORRECTION Patent N 3,733,25 Dated Inventor(s) It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column T, line 2 pasta should he eietr Column 7, Wine 19 sterpto" should be 22:32 2 "Jo'mmn 8, line 50 reqeuntly" shoufo he T qys ipir Columns 9 and 10 Table 5 "'Staphyloccus" should he EIEEDQlIKQ E QLQ.

line 2 under Composition of medium "glusoce" should he.

slwese Column 9 Table 1 "S.aureaus" should be Column 10, "line 7'5 Q pentahe diethyl ether" I should be;

mama is w ib r:

FORM PC4050 (10-59) USCOMM-DC 80376-P69 U 5, GQVERNMEHT PRlNYING OFFICE I 959 0*366'334.

UN TTD STATES PATENT oTTIcE TITITWHCATE F QORRECWON Patent No Z Dated Inven tor (s) It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Column 11, line 23 "ylene-benzene" should be ylene chloride-benzene column 15, line 25 "bottles in either" should be bottles on either Signed and sealed this 18th day of December 1973.

SEAT Attest:

EDWARD M.PLETCHER,JR. RENE D. TEGTMEYER Attesting Officer Acting Commissioner of Patents R Po'wso uscoMM-Dc eoanueea us. GOVERNMENT Pmm'me OFFICE: 1969 mass-3:44, 

